Hormones are chemical messengers that transport a signal from one cell to another. They play important roles in regulation of physiological activities. The effects of hormones include stimulation or inhibition of growth, regulating metabolism, induction or suppression of programmed cell death, and controlling the reproductive cycle, just to name a few. Thus, there are a wide range of clinical conditions that require frequent monitoring of hormones in biological samples, such as blood or tissue, for proper diagnosis and treatment of physiological disorders associated with the hormones.
For example, sex steroids are steroid hormones which are fundamental for growth and reproduction, and disturbances in their physiological levels can cause a multitude of clinical disorders, including growth retardation, infertility, or hormone-sensitive cancers (e.g., breast, endometrial and prostate cancers) [1-4]. Moreover, exogenous sex steroids have been used for decades as contraceptives, hormone replacements, and fertility and anti-cancer therapies [5-9]. Estrogen is a steroidal sex hormone of fundamental importance for normal human growth, reproduction and in breast cancer development and progression.
Diagnostic assays for the measurement of sex steroids are of central importance to the management of a wide range of clinical conditions and also play a key role in many areas of epidemiological study. Many clinical applications, such as the monitoring of blood levels of estradiol and other hormones in women undergoing treatment for infertility, have spurred the development of highly automated and cost-effective immunoassays.
Unfortunately, modern immunoassays often lack the low limit of detection required for some clinical areas, including monitoring menopausal women, patients on anti-estrogen therapies [10], or children [11-13]. Indeed, these clinical segments often present cases where the required assay dynamic range falls below the practical detection limits of commercial assays. This lack of performance in commercial assays has been demonstrated in studies of multiple automated analyzers, where considerable variability has been observed in results for samples with low concentrations. This failure of modern automated assays for sex steroids to address the clinical requirements in patient segments presenting low concentrations has prompted the Endocrinology Society to issue a statement cautioning against the use of standard assays for low level testosterone measurements. In recent years, a consensus has emerged that mass spectroscopy provides the optimal performance for low level steroid measurement.
Despite the widespread acknowledgement of the utility of mass spectroscopy as a preferred diagnostic tool low level steroid measurements, one of the key problems that limits its practical performance is sample preparation. Assays for steroids in clinical samples often require extensive pre-processing to extract the analytes from the matrix (i.e., unwanted chemical constituents and insoluble tissue debris). This is the case for all tissue samples, whole blood, and plasma and for any samples (including serum) that are to be evaluated by mass spectrometry or indirect immunoassays. Moreover, such processing techniques are known to be especially important for accurate quantification in subjects with low hormone levels. Unfortunately, sample processing techniques for analysis of steroids (including lysis, homogenization, extraction, and resolubilization) are labor-intensive (wasting many hours of laboratory time [14, 15]), and are thus prone to human error. Furthermore, assays often require hundreds of milligrams of tissue [16, 17] or milliliters of blood [18], which is ill-suited for routine clinical testing.
One promising technology for improved sample preparation is digital microfluidics. Digital microfluidics is a new technology in which discrete unit droplets are manipulated in air on the open surface of an array of electrodes by applying voltages to the electrodes [19]. Sample and reagent droplets with volumes of less than one microliter can be moved in multiple and reconfigurable paths defined by the actuation sequence of an array of electrodes. The mechanism for fluid motion depends on a host of factors and may be due to electrowetting or dielectrophoresis. Unlike conventional microfluidics, digital microfluidics enables the transport, mixing, merging and dispensing from reservoirs of single isolated droplets. The technology has been demonstrated for diverse applications including cell based assays, enzyme assays, protein profiling, and polymerase chain reaction [41].
Digital microfluidic arrays are typically made by depositing an array of electrodes onto a first planar surface and subsequently coating the surface with a layer of a hydrophobic insulator such as Teflon-AF. Electrical contact pads are connected to the electrodes, enabling the electrodes to be individually addressed. Sample and reagent reservoirs are incorporated into the array by including electrodes capable of supporting more than one unit droplet of fluid. Unit droplets can be extracted from reservoirs by actuating electrodes adjacent to the reservoir.
In a closed digital microfluidic array, a second planar surface, onto which a single planar electrode is typically deposited and subsequently coated with a layer of a hydrophobic insulator, is placed parallel to the first planar surface, forming a planar air gap. Accordingly, droplets are sandwiched between the two surfaces. Fluid droplets placed on the array elements (i.e. on the insulating layer directly above an array electrode) can be made to move by applying a voltage between an array electrode and the single planar electrode. Alternatively, an open digital microfluidic array can be formed with the first surface alone, where droplets are moved by applying a voltage between neighbouring electrodes.
While digital microfluidic systems have been successfully developed for a wide range of practical uses, the prior art has not provided a method of adapting their use to the efficient extraction of sex steroids or other hormones from biological samples. Thus, there is a need for a fast, economical and reliable method for analysis of hormone extraction and analysis in biological samples using digital microfluidics.